Purification of potato virus y for polyclonal antisera production and assessment of antisera specificity through DAS-ELISA

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Purified viral protein was obtained from Potato virus Y (PVY) pure culture maintained on potato (Solanum tuberosum L.) plants. The average concentration of the purified materials was found to be 153.1 ng/µl. PVY purified virus preparation was used for immunizing rabbit for production of polyclonal antisera against the virus. Good quality antisera were collected one week post boosters (AS4b, AS5b, AS6b and AS7b) and the IgG fractions were separated through ammonium sulphate precipitation. These four IgG fractions were tested for the detection of PVY by DAS-ELISA with universal anti- rabbit enzyme conjugate as secondary antibody, resulted high specificity with the known PVY infected samples.

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Nội dung Text: Purification of potato virus y for polyclonal antisera production and assessment of antisera specificity through DAS-ELISA

Int.J.Curr.Microbiol.App.Sci (2018) 7(8): 1311-1316 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 7 Number 08 (2018) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2018.708.148 Purification of Potato Virus Y for Polyclonal Antisera Production and Assessment of Antisera Specificity through DAS-ELISA Ranima Mishra1*, P.D. Nath1, B. Raigond2, R.C. Boro3, Shankar Hemanta Gogoi1 and Jutimala Phookan1 1 Department of Plant Pathology, 3Department of Agricultural Biotechnology, Assam Agricultural University, Jorhat 785013, Assam, India 2 Division of Plant Protection, ICAR- Central Potato Research Centre (CPRI), Shimla, Himachal Pradesh, India *Corresponding author ABSTRACT Purified viral protein was obtained from Potato virus Y (PVY) pure culture maintained on Keywords potato (Solanum tuberosum L.) plants. The average concentration of the purified materials was found to be 153.1 ng/µl. PVY purified virus preparation was used for immunizing DAS-ELISA, rabbit for production of polyclonal antisera against the virus. Good quality antisera were Purification, collected one week post boosters (AS4b, AS5b, AS6b and AS7b) and the IgG fractions Polyclonal antisera, were separated through ammonium sulphate precipitation. These four IgG fractions were Potato virus Y tested for the detection of PVY by DAS-ELISA with universal anti- rabbit enzyme Article Info conjugate as secondary antibody, resulted high specificity with the known PVY infected samples. The assay was compared with the commercial DAS-ELISA kit (Bio Reba, AG, Accepted: Switzerland). Results revealed that, among the antisera; AS6b was showing the highest 08 July 2018 mean absorbance values for all positive samples (2.210) and the value shown by the Available Online: commercial kit was slightly higher i.e., 2.250 and these were followed by AS5b (1.680), 10 August 2018 AS7b (0.929) and AS4b (0.362), respectively. The absorbance values shown by AS6b were statistically at par with the commercial kit. Introduction 2016) and Assam has marked its place among the top potato producing states within the Potato (Solanum tuberosum L., family: country; ranked seventh, with a total potato Solanaceae); recognized as the ‘king of production of 975.27 thousand tonnes. In vegetables’, had been cultivated in almost all Assam, 99.7 hectares of land is subjected to the states of India under diverse agro-climatic potato cultivation producing a total yield of conditions. India is an important potato 9.7 tonnes (Anon., 2018). producing country with an area estimated to be around 20.64 lakhs ha and production of Viral diseases were responsible for 455.99 lakh MT during 2015-16 (Anon., degeneration of potato cultivars, indicated by 1311
Int.J.Curr.Microbiol.App.Sci (2018) 7(8): 1311-1316 decrease in vigour, productivity of the crop samples collected from the district. The assay and resistance to diseases after successive was compared with PVY commercial DAS- cultivation from the same lot of tubers ELISA kit (Bio-Reba, AG, Switzerland) as (Nascimento et al., 2003). Potato virus Y control using the same healthy and PVY (PVY) was one of the most common and the infected plant samples. most dangerous virus found in potato, distributed world-wide, causing losses in the Materials and Methods form of reduced yield or quality of potato crop as well as seed tuber, which is an important Potato virus Y (PVY) isolates were maintained factor for yield of potato causing as much as in vitro on its host Potato (Solanum tuberosum 50 to 80 per cent yield loss in heavily infected L.). These plants were maintained in the green commercial lots (Hane and Hamm, 1999; house of the ICAR- Central Potato Research Nascimento et al., 2003; Biswas et al., 2005 Centre (CPRI), Shimla, Himachal Pradesh and Folwarczna et al., 2008). (Fig. 1). The leaves from those potato plants were used as the virus source for purification. Potato virus Y, the most dangerous virus in Purification was done according standard potato fields causing the disease common protocol (Khurana et al., 1987). Four hundred mosaic or potato severe mosaic had been one grams of leaves from those PVY infected of the most serious problems faced by potato potato plants were collected and kept in ice growers worldwide (Nolte et al., 2009). PVY water for one hour. Afterwards, leaves were was also reported as an important viral extracted in 0.1 M phosphate and sodium pathogen of potato crop in Jorhat district of buffer, pH-7.0 with 0.1% of 2- Assam with an incidence of 33.95 per cent mercaptoethanol, 0.1% DEECA and 0.015M (Mishra and Nath, 2015). of EDTA with ratio of 1:2. The sap was then transferred to 2ml microfuge tubes and As the virus affects tuber quality, therefore, centrifuged at 7800 rpm for 30 minutes. The production of disease free planting materials pellet was removed and equal volume of had been one of the most important factors for chloroform (CHCl3): carbon tetrachloride certification of potato tubers. Early detection (CCl4) (1:1 ratio) was added to the and identification of the virus in the field is supernatant. Afterwards, 40% polyethylene the need of the hour, for production of healthy glycol (PEG 6000) and 0.2 mol sodium crop lot. Therefore, efficient virus detection chloride (NaCl) were added and stirred at 4°C methods play an important role in producing for 60 minutes and incubate at 4°C for 60 and utilizing certified seed potatoes with minutes. This was then centrifuged at 7500 minimum virus infection and Enzyme linked rpm for 25 minutes and the supernatant was immunosorbent assay (ELISA) using specific collected. This was again centrifuged at 2500 antisera has been one of the most common for 10 minutes and the pellet was removed. method for the detection of potato viruses Afterwards, the supernatant was processed by (Sankari et al., 2007). adding 1% of triton-100 and stirred at 4°C for 120 minutes and precipitates at 10000 rpm for Hence, the present study was carried out 30 minutes. After removal of the supernatant, where we reported the production of the pellet was processed with the 0.01m polyclonal antisera against PVY in Jorhat sodium (Na) and potassium (K) phosphate district of Assam from North- East India for buffer, pH -7.0 with 0.5M urea and 0.1% 2- the first time and their specificity were mercaptoethanol (1/10) of the original volume confirmed by testing with PVY infected and kept at 4°C for overnight. Clarification 1312
Int.J.Curr.Microbiol.App.Sci (2018) 7(8): 1311-1316 was done at 10000 rpm for 10 minutes to (IgG batches from booster injections) and the remove the pellet. The supernatant was PVY commercial IgG for their specificity in processed to centrifuge on sucrose cushion the DAS- ELISA assay with healthy and PVY 30% (W/V) (density gradient) in 0.01M infected leaf extracts. sodium (Na) and potassium (K) phosphate buffer pH -7.0 (15ml of sample for 5ml Results and Discussion sucrose at 30%) at 25000 rpm for 150 minutes in ultra-centrifuge to get the viral protein as The purification was done in two batches. The pellet. Titre of purified viral suspension was absorbance values of the two batches of measured through nanodrop quantification. purified material were found as 1.19 and 1.38, respectively, at 260 nm, while the absorbency PVY purified virus preparation was used for at 280 nm were found as 1.02 and 1.07 for immunizing rabbit for production of antisera. both the batches, respectively. Rabbit was immunized subcutaneously, similar to Nath (2000), at two weeks interval The average concentration of the purified for the first three injections and after these; materials was found to be 153.1 ng/µl. four boosters were given at six weeks intervals. Bleeds were taken one week after The antisera recovered following the first each injection. Immunoglobulins (IgG) were three injections showed very low absorbance purified by the ammonium sulphate values and hence, were not considered for precipitation and titres were measured through further serodiagnostic assays. Among the nanodrop quantification. The antisera from the antisera from the booster doses, AS6b booster injections were designated as AS4b, (antisera obtained after third booster injection) AS5b, AS6b and AS7b. showed the highest absorbance value; viz., 1.44 mg/ml at the wave length of 280 nm, 26 IgG from the booster antisera were tested for weeks after first injection which was followed their sensitivity and specificity against known by AS5b, AS7b and AS4b (Fig. 3). PVY healthy and infected plant samples in DAS- ELISA and the assay was compared The four batches of antisera, from the booster with PVY commercial DAS- ELISA kit (Bio- injections, viz.; AS4b, AS5b, AS6b and AS7b, Reba, AG, Switzerland) as control using the were tested for their sensitivity against PVY in same healthy and PVY infected plant samples. DAS- ELISA assay with five replications DAS- ELISA was carried out as per standard maintaining both primary and secondary protocol (Fig. 2) and the antisera (as primary antibody dilutions at 10-3. The assay was IgG) were used with the PVY antibody- compared with PVY commercial DAS- alkaline phosphatase conjugate from Bio ELISA kit (Bio-Reba, AG, Switzerland) as Reba, AG, Switzerland; in the assay. The control using the same healthy and PVY assay was quantitative, replicated five times infected plant samples. All the antisera could and every time saps from both healthy and successfully detect PVY in the potato leaf infected plants were taken in the study. extracts. Among the produced antisera AS6b was showing the higher absorbance values for Statistical analysis the leaf extracts which were at par with the PVY commercial IgG from the kit followed Completely randomized design (CRD) by AS5b, AS7b and AS4b. These results analysis was done to calculate out the critical reflected high sensitivity as well as specificity difference (CD) among the tested antisera of the antisera batches against PVY. 1313
Int.J.Curr.Microbiol.App.Sci (2018) 7(8): 1311-1316 Fig.1 Potato virus Y (PVY) isolates maintained in vitro on its host plant Potato (Solanum tuberosum L.). (a) PVY infected potato plants maintained as virus source in the green house (b) collection of leaf samples from PVY infected potato plants for virus purification Fig.2 DAS- ELISA assay with produced antisera and comparison with PVY commercial kit. (a) sensitization of plate with addition of IgG, (b) addition of homogenized sample, (c) addition of IgG conjugated, (d) IgG enzyme- conjugate giving a colour reaction in presence of its substrate Fig.3 Absorbance (OD280) values for PVY antisera batches from booster injections in nanodrop quantification 1314
Int.J.Curr.Microbiol.App.Sci (2018) 7(8): 1311-1316 Table.1 DAS- ELISA values for leaf extracts of potato samples Antisera ELISA value for Minimum Maximum Mean ELISA for all positive control ELISA value ELISA positive samples value AS4b 0.077 0.097 0.727 0.362 AS5b 0.512 1.062 1. 950 1.680 AS6b 0.740 1.480 2.850 2.210 AS7b 0.303 0.520 1.776 0.929 PVY 0.991 1.592 2.990 2.250 (commercial kit) CD (P= 0.05) 0.236 Table.2 Duncan’s Multiple Range Test (DMRT) between antisera batches and PVY (Commercial kit) DAS- ELISA values IgG Subset AS4b AS7b AS5b AS6b PVY (Commercial kit) AS4b 0.348d AS7b 0.898c AS5b 1.626b AS6b 2.144a PVY 2.193a (Commercial kit) Significance 1.000 1.000 1.000 1.000 1.000 *Means for groups in homogeneous subsets are displayed The absorbance values (OD405) for extracts of calculated out by completely randomized potato leaf tissues were recorded by design (CRD) analysis for their specificity comparing the absorbance values for negative against PVY. The absorbance values shown control and the positive control. A value by AS6b were statistically at par with the which was five times higher than the average commercial kit (Table 2). These results of negative controls, considered as positive. reflected high sensitivity as well as specificity Results revealed that, among the antisera; of the antisera batches against PVY. AS6b was showing the highest mean absorbance values for all positive samples All the four antisera batches from the booster (2.210) and the value shown by the injections showed highly sensitive and commercial kit for the same was slightly specific reaction against PVY in DAS- higher; i.e., 2.250. These were followed by ELISA assay. AS6b showed the highest AS5b (1.680), AS7b (0.929) and AS4b sensitivity with the maximum DAS- ELISA (0.362), respectively (Table 1). value followed by AS5b and AS7b respectively compared with the commercial Critical difference (CD) among the four IgG kit. High quality antisera could be produced batches and PVY commercial IgG was through rabbit immunizations against PVY. 1315
Int.J.Curr.Microbiol.App.Sci (2018) 7(8): 1311-1316 This can be carried out for different viruses of two potato cultivars expressing mild quarantine importance which will further help symptoms. Plant Dis. 83: 43- 45. in early detection and identification, Khurana SMP, Singh MN and Kumar S. certification of disease free planting material 1987. Purification of Potato Virus Y as, early detection helps in management at and the production of antiserum. Curr appropriate time. Sci. 56: 420- 422. Mishra R and Nath PD. 2015. Incidence and References detection of Potato virus Y on potato crop in Jorhat district of Assam by Anonymous. (2016). A brief report on potato DAS-ELISA. Indian Phytopath. 68 (4): production in the country. National 467- 469. Horticultural Research and Nascimento LC, Pio-Riberio G, Willadino L Development Foundation. http://nhrdf. and Andrade GP. 2003. Stock indexing org/pdf/POTATO%20CROP%20REPO and Potato Virus Y elimination from RT- %2008%20APRIL %20 2016.pdf. potato plants cultivated in vitro. Scientia Retrieved on 18.04.2018. Agricola. 60: 525- 530. Anonymous. (2018). Top 10 largest potato Nath, P.D. (2000). Studies on Rice Tungro producing states in India in 2018. disease with special reference to www.thedailyrecords.com. Retrieved on purification, serology and molecular 18.04.2018. mapping of resistance to Rice Tungro Biswas M K, De BK and Nath PS. 2005. Rate Spherical Virus. Ph. D. thesis submitted of spread of PVX, PVY and PLRV to Assam Agricultural University. diseases to potato varieties. Ann Plant Assam. Prot Sci. 13:165-178. Nolte, P., Alvarez, J.M. and Whitworth, J.L. Folwarczna J, Plchova H, Moravec T, 2009. Potato virus Y management for Hoffmeisterova H, Dedic P, Cerovska the seed potato producer. Extension N. 2008. Production of polyclonal Bulletin. University of Idaho. pp. 1-8. antibodies to a recombinant coat protein Sankari S, Ali MC and Katayama K et al., of Potato virus Y. Folia Microbiol. 53 2007. The first report of polyclonal (5): 438- 442. antibody production of a Syrian isolate Hane DC and Hamm PB. 1999. Effects of of Potato virus Y. J Agric Sci. 52 (2): seed borne Potato virus Y infection in 109- 114. How to cite this article: Ranima Mishra, P.D. Nath, B. Raigond, R.C. Boro, Shankar Hemanta Gogoi and Jutimala Phookan. 2018. Purification of Potato Virus Y for Polyclonal Antisera Production and Assessment of Antisera Specificity through DAS-ELISA. Int.J.Curr.Microbiol.App.Sci. 7(08): 1311-1316. doi: https://doi.org/10.20546/ijcmas.2018.708.148 1316